Bacteria of the genus Salmonella are notorious for their pathogenicity in both man and animals. In the USA alone, on a yearly basis the number of humans suffering from Salmonella infections exceeds the two million cases. In most cases, the infection is caused by contaminated food. Well-known sources of infection are eggs (from both ducks and chickens), products containing eggs and not sufficiently heated poultry and pig meat. Especially in infants, young children, elderly people and immune compromised patients, the ability to cope with such infections is low. In these groups, the yearly death rate due to Salmonella infections is high. During the last few decades the more efficient large-scale animal husbandry has led to an enormous increase in animal density. As a result, an increase is seen of the number of animal infections and subsequently in human infections caused by infected food. It is clear that animals are the main source of Salmonella infection. This source is very difficult to control. First of all Salmonella infections in most cases cause no serious illness in healthy full-grown animals; these animals can carry the bacterium for a prolonged period. During that time they are shedding the bacterium in their dung. This makes it practically impossible to avoid infection in the more vulnerable young animals. Secondly, many Salmonella species colonise several different host species. Some of the Salmonella species cause primary infections in specific hosts, whereas other Salmonella species are not restrictive at all. As a primary infectans, S. typhi and paratyphi are frequently associated with infection in man. S. dublin is connected with cattle, S. abortus-equi causes abortion in horses. S. abortus-ovi causes abortion in sheep. S. choleraesuis is the cause of lethal diarrhoea in young pigs. S. typhimurium and S. enteritidis cause salmonellosis in humans, poultry, pigs, cattle and rodents, S. arizonae causes disease in turkeys, whereas S. gallinarum causes salmonellosis only in poultry.
It is clear that there is a need for good, safe and efficacious vaccines for combating the various Salmonella species. Currently, several live attenuated Salmonella vaccines are commercially available.
Many of the strains that are suitable for use in a live vaccine are attenuated to a level that makes them virtually non-virulent. Two striking examples of such non-virulent strains are the Salmonella gallinarum 9R strain and the Salmonella typhimurium SL3261 strain.
The Salmonella gallinarum 9R strain was described as long as 44 years ago by Smith (J. Hyg. Camb., 54:419–432 (1956)). This highly attenuated strain is known to have at least a mutation in a gene involved in the synthesis of the cell's O-polysaccharides, leading to a Smooth→Rough mutation. The 9R strain has been used as a Rough reference strain since then (see e.g. Cameron, C. M. et al. , Onderstepoort J. Vet. Res. 39(3), 139–146 (1972)). This strain can be administered to chicken even in a dose of 109 bacteria without causing the death of any chicken, whereas a dose comprising 103 wild-type bacteria kills all animals.
The Salmonella typhimurium SL3261 strain has been described 19 years ago by Hoiseth, S. and Stocker, B. A. D. (Nature 291: 238–239(1981)) and is available from Deposit number SGSC 439, Salmonella Genetic Stock Centre, University of Calgary, Alberta, Canada T2N 1N4.
This strain is known to have a highly attenuating mutation in the aromatic pathway synthesis. This strain can be administered even in a 106 bacteria dose to susceptible mice without causing the death of any animal, whereas the LD50 of the parent strain for these mice is less then 20 bacteria.
Such Salmonella strains have since long been appreciated for their highly attenuated character. They are so severely attenuated that they are described in the literature as non-virulent. Therefore they have been the strains of choice for live attenuated Salmonella vaccines.
In principle, there is a clear relation between the level of virulence and the level of immunity induced. In general the strains with the highest virulence induce the highest levels of immunity: animals that survive infections with wild-type Salmonella strains often build up a long-lasting immunity. On the other hand, for use in vaccines those strains that induce no pathogenesis at all, the non-virulent strains, are the most desirable but such strains are often not capable of inducing a sufficiently high level of immunity. The non-virulent Salmonella strains described above are just about capable of triggering the immune system to a sufficient level.
A relevant disadvantage of all live attenuated vaccines is however, that in principle they can revert to a wild-type level of virulence through recombination of the mutated gene with DNA from bacteria that do carry non-mutated genes, such as e.g. wild-type field strains. Such a recombination event can take place in various ways, e.g. through transfection, transduction or transformation. Genes that are known to play a role in these recombination processes are known as rec-genes. One of the rec-genes of key importance is the recA-gene. This gene encodes an enzyme RecA that is involved in several steps of the recombination process. recA and many other rec-genes have been described i.a. by Lloyd. R. G. and Low, K. B. (“Escherichia coli and Salmonella”, sec. ed., ASM Press, ISBN 1-55581-0-845, par. 119 page 2236–2255), by West, S. C. (Annu. Rev. Biochem. 61: 603–640 (1992), and by Kowalczykowski. S. C. et al (Microbiol Rev. 58: 401–465 (1994)).
At first sight it seems therefore tempting, when contemplating a safe live attenuated vaccine, to delete one of the rec-genes. Such a deletion severely impairs the ability of the bacterial DNA to recombine. Nevertheless, deletion of rec-genes is also known to cause severe attenuation of the bacterial strains from which it has been deleted. Deletion of e.g. the recA- or the recBC-genes in Salmonella makes the mutants sensitive to the oxidative burst of macrophages, which leads i.a. to significant in vivo growth suppression. This has been demonstrated for Salmonella, e.g. by Buchmeier, N. A. et al., (Mol. Microbiol, 7:933–936 (1993)) and equally convincing for other bacterial genera as distantly related to Salmonella as e.g. Vibrio cholerae (Ketley et al., Infect. and Immun. 58: 1481–1484 (1990)).
Thus, where deletion of a rec-gene might be an advantage for virulent strains, it certainly would be assumed to be a disadvantage for non-virulent strains, such as the Salmonella gallinarum 9R strain and the Salmonella typhimurium SL3261 strain. These strains, known to be already highly impaired, could not be expected to trigger any immunological response at all after removal of rec-genes. This may explain why no attempts were made to delete Rec-genes from such already highly attenuated, non-virulent strains as S. gallinarum 9R and S. typhimurium SL3261, in spite of their long history.